National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi, 214112, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Microb Cell Fact. 2020 May 26;19(1):113. doi: 10.1186/s12934-020-01370-9.
Corynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum.
In this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L.
In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.
谷氨酸棒杆菌是一种传统的食品级工业微生物，近年来正在开发一种高效的无内毒素重组蛋白表达工厂。然而，其重组蛋白表达水平低的固有缺点仍然难以解决。在这里，根据细菌特异性的多顺反子特征，即多个蛋白质可以在一个 mRNA 中翻译，我们努力在上游插入一个前导肽基因，作为目标基因的表达增强子，结果发现，这可以显著提高在诱导型 tac 启动子控制下的蛋白质的表达水平。
在本研究中，构建了含有 24 种不同前导肽序列的大肠杆菌（E. coli） tac 启动子，在谷氨酸棒杆菌中以双顺反子的形式构建。分离出了三种强双顺反子表达载体，在不同的培养条件下均表现出高效。进一步使用六种模型蛋白（醛脱氢酶（ALDH）、醇脱氢酶（ADH）、RamA（乙酸代谢调节剂）、牛干扰素-α（BoIFN-α）、传染性牛鼻气管炎病毒（IBRV）糖蛋白 D 蛋白（gD）和前胶原类型Ⅰ N 端肽（PΙNP））验证了这些双顺反子载体的兼容性。与原始带有 tac 启动子的载体相比，所有检测到的蛋白均有较高的表达。在分批补料培养中也进行了 PΙNP 的大规模生产，最高 PΙNP 产量为 1.2g/L。
本研究通过筛选和在目标基因前插入前导肽序列，提高了谷氨酸棒杆菌诱导型 tac 启动子的强度。具有双顺反子表达模式的载体具有很强的兼容性，能够高效表达各种异源蛋白。这种新策略可用于进一步提高诱导型启动子的性能，实现诱导控制和高产的双重能力。
